Novel bee venom polypeptides and methods of use thereof

ABSTRACT

The present invention provides novel protein named Api m 6. Api m 6 was identified in bee venom and is found in four isoforms, each about 8 kD. The purified Api m 6 can be used to modulate immune responses, e.g., in individuals hypersensitive to bee venom.

FIELD OF THE INVENTION

[0001] The present invention relates to novel polypeptides derived frombee venom and methods of use thereof, including pharmaceuticalcompositions useful for modulating immune responses to bee pollen.

BACKGROUND OF THE INVENTION

[0002] Bee venom (BV) is a complex mixture of antigens that can includeone or more toxic polypeptides. Many of these polypeptides arehypersensitizing agents and can additionally have hemolytic orneurotoxic effects.

[0003] Some individuals are hypersensitive to BV polypeptides. IgEantibodies from BV-hypersensitive individuals recognize several BV toxicpolypeptides. BV polypeptides, often referred to as allergens,recognized by IgE in BV hypersensitive individuals can include, e.g.phospholipase A₂ (PLA₂), acid phosphatase, hyaluronidase, allergen C,and other, high molecular weight (MW) proteins.

[0004] BV hypersensitive individuals can be at high risk of an adversereaction to a bee sting. One recognized method for preventing orminimizing serious adverse reactions resulting from a bee sting is todesensitize the individual to the allergens present in BV. Thisprotection can be induced by a process termed venom immunotherapy (VIT).

SUMMARY OF THE INVENTION

[0005] The invention is based, in part, on the discovery of a novel beevenom protein, which has been named Api m 6. Polypeptides derived fromApi m 6 polypeptides can be used, e.g., in venom immunotherapy toprotect susceptible individuals from the adverse effects of a bee sting.

[0006] Accordingly, in one aspect, the invention provides asubstantially pure polypeptide comprising an amino acid sequence atleast 70% identical, and more preferably at least 90% identical, to theamino acid sequence of an Api m 6 polypeptide, e.g., a peptide whichincludes the amino acid sequence of SEQ ID NO:1. In some embodiments,the polypeptide binds to a human IgE antibody.

[0007] In other embodiments, the polypeptide includes a sequence atleast 70% identical to one or more of the amino acid sequences of SEQ IDNOs: 2-4. Preferably, the polypeptide has an amino acid sequencecorresponding to the amino acid sequence of an Apis spp. bee venomprotein, e.g., an Apis mellifera bee venom protein. In some embodiments,the polypeptide is glycosylated.

[0008] The polypeptide may, in some embodiments, stimulate T-cellproliferation.

[0009] Preferably, the polypeptide binds to the monoclonal antibody 5E11(Accession No.______).

[0010] The invention also features an antibody that binds to an Api m 6protein. The antibody can be a polyclonal or a monoclonal antibody. Insome embodiments, the antibody binds to the same epitope to which themonoclonal antibody produced by the hybridoma 5E11 (Accession No.______)binds. In preferred embodiments, the antibody is the monoclonal antibodyproduced by the hybridoma 5E11 (Accession No.______). The invention alsoincludes a hybridoma producing an antibody which binds to the sameepitope to which the monoclonal antibody produced by the 5E11 (AccessionNo.______). Preferably, the hybdridoma is the hybridoma 5E11 (AccessionNo.______).

[0011] The invention also features a composition comprising polypeptidefragments of the Api m 6 protein, wherein the polypeptide fragments arebetween 6-72 amino acids in length. In preferred embodiments, thepolypeptide fragments are between 20-100, 30-70, or 40-60 amino acids inlength. Preferably, at least one polypeptide in the composition has anamino acid sequence that overlaps by at least 3 amino acids with atleast one other polypeptide in the composition, e.g., polypeptidefragments of Api m 6 that overlap by between 5 and 10 amino acids. Inthe most preferred embodiments, the composition comprises of a set ofpolypeptide fragments that map the total length of the Api m 6 protein.

[0012] The invention also features a pharmaceutical composition thatincludes an Api m 6 polypeptide and a pharmaceutically acceptablecarrier. In some embodiments, the pharmaceutical composition includes anadditional polypeptide, e.g., a second, third, fourth, or more bee venompolypeptide or polypeptides.

[0013] The additional bee venom polypeptides can include, e.g.,phospholipase A₂, hyaluronidase, allergen C, mellitin, adolapin,minimine, acid phosphatase, protease inhibitor, and glycosylatedIgE-binding proteins, or analogs or derivatives thereof.

[0014] In another aspect, the invention features a method of modulatingan immune response. The method includes administering an Api m 6polypeptide to a subject in need thereof in an amount sufficient toinhibit an immune reaction by the subject against the polypeptide. Ifdesired, one or more additional bee venom polypeptides may also beadministered to the subject. The additional bee venom polypeptides caninclude, e.g., phospholipase A₂, hyaluronidase, allergen C, mellitin,adolapin, minimine, acid phosphatase, protease inhibitor, andglycosylated IgE-binding proteins, or analogs or derivatives thereof.

[0015] In a further aspect, the invention includes a method ofidentifying an individual at risk for bee venom hypersensitivity. Themethod includes administering to the individual an Api m 6 polypeptideand measuring an immune response raised against the polypeptide. Adetectable immune response indicates that the individual is at risk forbee venom hypersensitivity. In preferred embodiments, the Api m 6polypeptide is administered intradermally. Preferably, the Api m 6polypeptide is administered at a concentration of less than about 1μg/ml.

[0016] Also provided is a method of purifying an Api m 6 polypeptide.The method includes providing a cell expressing the Api m 6 polypeptide.The cell is then contacted with an antibody which binds to a polypeptidethat includes an amino acid sequence at least 70% identical to the aminoacid sequence of SEQ ID NO:1 under conditions which allow for formationof a polypeptide-antibody complex. The antibody-polypeptide complex isthen isolated, and the Api m 6 polypeptide is recovered from thecomplex.

[0017] Also provided by the invention is a kit that includes, in one ormore containers, an Api m 6 polypeptide, overlapping polypeptidefragments of an Api m 6 polypeptide, an anti Api m 6 polypeptideantibody (e.g., a monoclonal or polyclonal antibody), or a combinationof these polypeptides or antibodies.

[0018] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

[0019] Other features and advantages of the invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWING

[0020]FIG. 1 is a schematic representation of the Api m 6 isoforms. Theorder of the amino acids in brackets was not determined.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The invention provides a novel 8 kD bee venom protein, termed Apim 6, that was identified based on its reactivity to IgE antisera fromindividuals who are hypersensitive to bee venom. Four isoforms of theApi m 6 polypeptide have been identified. These are: Api m 6.01, whichincludes the amino acid sequence shown in SEQ ID NO:1 and has apredicted molecular weight of 7,190 Da; Api m 6.02 Da, which includesthe amino acid sequence of SEQ ID NO:2 and has a predicted molecularweight of 7,400; Api m 6.03 Da, which includes the amino acid sequenceof SEQ ID NO:3 and has a predicted molecular weight of 7,598 Da; and Apim 6.04, which has the amino acid sequence of SEQ ID NO:4 and has amolecular weight of 7,808 Da.

[0022] The four isoforms are present in approximately equimolar amounts.The isoforms share a common central amino acid sequence of 67 residuesand differ only in their amino and carboxyl terminus by up to 6 aminoacids (FIG. 1). The common 67 amino acid core sequence is shown as Api m6.01 (SEQ ID NO:1). Api m 6.03 (SEQ ID NO:3) and Api m 6.04 (SEQ IDNO:4) have an additional N-terminal “Phe-Gly-Gly-Phe” relative to bothApi m 6.01 (SEQ ID NO:1) and Api m 6.02 (SEQ ID NO:2). Furthermore, Apim 6.02 and Api m 6.04 have two additional residues, Pro and Leu, at theC-termimus. The relative order of these amino acids has not yet beendetermined.

[0023] This novel Api m 6 protein is useful as an allergen forimmunotherapy. Development of a novel bee venom immunotherapy strategybased on overlapping peptides is also described.

[0024] Sequences and Corresponding SEQ ID Numbers

[0025] The sequences and corresponding SEQ ID NOs discussed hereininclude the following: SEQ ID NO:1 Api m 6.01 (7,190 Da) amino acidsequence (67 aa) SEQ ID NO:2 Api m 6.02 (7,400 Da) amino acid sequence(69 aa) SEQ ID NO:3 Api m 6.03 (7,598 Da) amino acid sequence (71 aa)SEQ ID NO:4 Api m 6.04 (7,808 Da) amino acid sequence (73 aa)

[0026] Api m 6 Polypeptides

[0027] One aspect of the invention pertains to isolated Api m 6polypeptides and proteins, including variants such as biologicallyactive portions thereof. In one embodiment, native Api m 6 proteins canbe isolated by an appropriate purification scheme using standard proteinpurification techniques. Alternatively, an Api m 6 protein orpolypeptide can be synthesized chemically using standard peptidesynthesis techniques, or can be produced by recombinant DNA techniques.

[0028] An “isolated” or “purified” protein or biologically activeportion thereof is substantially free of material (e.g., other,contaminating proteins) from the cell suspension, tissue source, orvenom preparation from which the Api m 6 protein is derived, orsubstantially free from chemical precursors or other chemicals whenchemically synthesized. The language “substantially free of othermaterial” includes preparations of Api m 6 protein in which the proteinis separated from cellular components of the cells from which it isisolated or recombinantly produced. In one embodiment, the language“substantially free of other material” includes preparations of Api m 6protein having less than about 30% (by dry weight) of non-Api m 6protein (also referred to herein as a “contaminating protein”), morepreferably less than about 20% of non-Api m 6 protein, still morepreferably less than about 10% of non-Api m 6 protein, and mostpreferably less than about 5% non-Api m 6 protein. When the Api m 6protein or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume of the protein preparation.

[0029] The language “substantially free of chemical precursors or otherchemicals” includes preparations of Api m 6 protein in which the proteinis separated from chemical precursors or other chemicals which areinvolved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of Api m 6 protein having less than about 30% (bydry weight) of chemical precursors or non-Api m 6 chemicals, morepreferably less than about 20% chemical precursors or non-Api m 6chemicals, still more preferably less than about 10% chemical precursorsor non-Api m 6 chemicals, and most preferably less than about 5%chemical precursors or non-Api m 6 chemicals.

[0030] “A polypeptide having a biologically active portion of Api m 6”refers to polypeptides exhibiting activity similar, but not necessarilyidentical to, activity of a polypeptide of the present invention,including mature forms, as measured in a particular biological assay,with or without dose dependency. Biologically active portions of an Apim 6 protein include peptides comprising amino acid sequencessufficiently homologous to or derived from the amino acid sequence ofthe Api m 6 protein, e.g., the amino acid sequence shown in any of SEQID NOs: 1-4, which include fewer amino acids than the full length Api m6 proteins, and exhibit at least one activity of an Api m 6 protein,e.g., the ability to stimulate T-cell proliferation or the ability tobind IgE antibody from, e.g., an individual hypersensitive to bee venom.Typically, biologically active portions comprise a domain or motif withat least one activity of the Api m 6 protein. A biologically activeportion of an Api m 6 protein can be a polypeptide which is, forexample, 10, 15, 25, 35, 45, 55, 60, or 65 or more amino acids inlength.

[0031] In some embodiments, the Api m 6 protein has an amino acidsequence shown in any of SEQ ID NOs: 1-4. Preferably, the Api m 6protein has the amino acid sequence of a protein isolated from bee venomfrom an Apis spp., e.g., Apis mellifera.

[0032] In other embodiments, the Api m 6 protein is substantiallyhomologous to the sequences shown in SEQ ID NO:1-4 and retains thefunctional activity of the protein of SEQ ID NOs:1-4, yet differs inamino acid sequence due to natural allelic variation or mutagenesis, asdescribed in detail below. Accordingly, in other embodiments, the Api m6 protein is a protein which comprises an amino acid sequence at leastabout 45%, 55%, 65%, 75%, 85%, 90%, 95%, 97%, 98%, or even 99%homologous to any of SEQ ID NOs: 1-4, or a fragment thereof, e.g. afragment having one or more activities of an Api m 6 protein.

[0033] When peptides derived the Api m 6 proteins or variants describedherein are used to tolerize an individual sensitive to a proteinallergen, e.g., by subcutaneous administration, the peptide ispreferably derived from a protein allergen of the genus Apis. Longoverlapping peptides comprising at least one epitope of the Apisallergen phospholipase A₂ have been described. See, e.g., Kammerer, etal., Clin and Exp Allergy 27: 1016-1026 (1997) and Kammerer, et al., JAllergy Clin Immunol 100: 96-103 (1997).

[0034] Various Api m 6 proteins, as well as derivatives, fragments andanalogs thereof, can be screened for the ability to alter and/ormodulate cellular functions. This includes those relating to modulatingimmune response, e.g., T-cell proliferation and IgE-mediated immunereactions. The derivatives, fragments or analogs that possess thedesired immunogenicity and/or antigenicity may be utilized inimmunoassays, for immunization, for inhibition of the activity of anaforementioned peptide, etc. For example, derivatives, fragments oranalogs that retain (or, alternatively lack or inhibit) a given propertyof interest may be utilized as inducers (or inhibitors) of such aproperty and its physiological correlates. Derivatives, fragments andanalogs of an aforementioned peptide may be analyzed for the desiredactivity or activities by procedures known within the art.

[0035] Manipulations of the sequences included within the scope of theinvention may be made at the peptide level. Included within the scope ofthe present invention is an aforementioned peptide (or a fragment,derivative, or analog thereof) that is differentially modified during orafter translation or synthesis (e.g., by glycosylation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to an antibody molecule or othercellular ligand, and the like). Any of the numerous chemicalmodification methods known within the art may be utilized including, butnot limited to, specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH₄, acetylation, formylation,oxidation, reduction, metabolic synthesis in the presence oftunicamycin, etc. In a specific embodiment, sequences of anaforementioned peptide are modified to include a fluorescent label. Inanother specific embodiment, an aforementioned peptide is modified bythe incorporation of a heterofunctional reagent, wherein suchheterofunctional reagent may be used to cross-link the members of acomplex.

[0036] Complexes of analogs and derivatives of the Api m 6 proteins andvariants can be chemically synthesized. For example, a peptidecorresponding to a portion of an aforementioned peptide that comprises adesired domain or that mediates a desired activity in vitro, may besynthesized by use of a peptide synthesizer. In cases where naturalproducts are suspected of being mutant or are isolated from new species,the amino acid sequence of an aforementioned protein isolated from thenatural source, may be determined, e.g., by direct sequencing of theisolated protein. The peptides may also be analyzed by hydrophilicityanalysis (see, e.g., Hopp and Woods, Proc Natl Acad Sci USA 78:3824-3828 (1981)) which can be used to identify the hydrophobic andhydrophilic regions of the peptides, thus aiding in the design ofsubstrates for experimental manipulation, such as in bindingexperiments, antibody synthesis, etc. Secondary structural analysis mayalso be performed to identify regions of a peptide that adopt specificstructural motifs. See e.g., Chou and Fasman, Biochem 13: 222-223(1974). Manipulation, translation, secondary structure prediction,hydrophilicity and hydrophobicity profiles, open reading frameprediction and plotting, and determination of sequence homologies, canbe accomplished using computer software programs available in the art.Other methods of structural analysis including, but not limited to,X-ray crystallography (see, e.g., Engstrom, Biochem Exp Biol 11: 7-13(1974)); mass spectroscopy and gas chromatography (see, e.g., Methods inProtein Science, 1997. J. Wiley and Sons, New York, N.Y.) and computermodeling (see, e.g., Fletterick and Zoller, eds., 1986. ComputerGraphics and Molecular Modeling, In: Current Communications in MolecularBiology, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)may also be used.

[0037] In some embodiments, one or more Api m 6 peptides are present ina composition in which one Api m 6 peptide overlaps by at least 3 aminoacids with at least one other Api m 6 polypeptide in the composition. Inmost preferred embodiments, the peptides overlap between 5 and 10 aminoacids. In certain embodiments, a composition used for tolerizationincludes a set of polypeptide fragments that map the entire length ofthe Api m 6 protein. In an additional embodiment, the amino acidsequences of one or more peptides can be produced and joined by a linkerto increase sensitivity to processing by antigen-presenting cells. Suchlinker can be any non-epitope amino acid sequence or other appropriatelinking or joining agent.

[0038] Api m 6 proteins having altered amino acid sequences can beconstructed, e.g., by chemical synthesis techniques. In someembodiments, the changes do not alter the functions of the variantproteins relative to the starting Api m 6 protein, e.g., an Api m 6protein having the amino acid sequence of any of SEQ ID NOs: 1-4. Aminoacid substitutions are preferably at “non-essential” amino acidresidues. A “non-essential” amino acid residue is a residue that can bealtered from the wild-type sequence of Api m 6 (e.g., the sequence ofany of SEQ ID NOs: 1-4) without altering the biological activity,whereas an “essential” amino acid residue is required for biologicalactivity. For example, amino acid residues that are conserved among theApi M 6 proteins from different species, e.g., different Apis spp., arepredicted to be particularly unamenable to alteration.

[0039] The invention also includes variant Api m 6 proteins that containchanges in amino acid residues that are not essential for activity. SuchApi m 6 proteins differ in amino acid sequence from any or all of SEQ IDNOs:1-4, yet retain biological activity. The variant Api m 6 proteinsinclude in which one or more amino acid substitutions, additions ordeletions are introduced into the protein.

[0040] Preferably, conservative amino acid substitutions are made at oneor more predicted non-essential amino acid residues. A “conservativeamino acid substitution” is one in which the amino acid residue isreplaced with an amino acid residue having a similar side chain.Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted non-essential amino acid residue in Api m 6 is preferablyreplaced with another amino acid residue from the same side chainfamily. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of an Api m 6 coding sequence, toidentify mutants that retain activity.

[0041] In some embodiments, a mutant Api m 6 protein can be assayed for(1) the ability to form protein:protein interactions with Api m 6proteins, other proteins, or biologically active portions thereof, (2)complex formation between a mutant Api m 6 protein and an Api m 6ligand; (3) the ability to stimulate T-cell proliferation; or (4) theability to bind IgE antibodies from, e.g., the sera of an individualhypersensitive to bee venom.

[0042] A peptide derived from a protein allergen can be tested todetermine whether the peptide binds IgE specific for the allergen andresult in the release of mediators (i.e. histamine) from mast-cells orbasophils.

[0043] T-cell stimulating activity can be tested by culturing T-cellsobtained from an individual sensitive to the Api m 6 proteins andvariants described herein (i.e., an individual who has an immuneresponse to the protein allergen or protein antigen) with an Api m 6protein or variant and determining the presence or absence ofproliferation by the T-cells in response to the peptide as measured by,for example, uptake of tritiated thymidine. Stimulation indicies forresponses by T-cells to peptides useful in methods of the invention canbe calculated as the maximum counts per minute (CPM) taken up inresponse to the peptide divided by the CPM of the control medium. Forexample, a peptide derived from a protein allergen may have astimulation index of about 2.0. A stimulation index of at least 2.0 isgenerally considered positive for purposes of defining peptides usefulas immunotherapeutic agents. Preferred peptides have a stimulation indexof at least 2.5, more preferably at least 3.5 and most preferably atleast 5.0.

[0044] Also included in the invention are fragments of a protein havinga portion of the amino acid sequence of any of SEQ ID NOs:1-4. Thefragment preferably has one or more of the herein described activitiesof an Api m 6 protein. The fragment can be, e.g., 6-72, 20-90, 30-70, or40-60 amino acids in length.

[0045] To determine the percent homology of two amino acid sequences orof two nucleic acids, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoor nucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are homologous at that position(i.e., as used herein amino acid or nucleic acid “homology” isequivalent to amino acid or nucleic acid “identity”). The percenthomology between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e., percent homologyequals the number of identical positions divided by the total number ofpositions times 100).

[0046] The Api m 6 proteins, fragments, and other variants describedherein can be modified. Thus, the invention includes, e.g.,myristylated, glycosylated, and phosphorylated Api m 6 proteins andtheir derivatives.

[0047] The invention also provides Api m 6 chimeric or fusion proteins.As used herein, an Api m 6 “chimeric protein” or “fusion protein”comprises, an Api m 6 polypeptide operatively linked to a non-Api m 6polypeptide. A “Api m 6 polypeptide” refers to a polypeptide having anamino acid sequence corresponding to Api m 6, whereas a “non-Api m 6polypeptide” refers to a polypeptide having an amino acid sequencecorresponding to a protein which is not substantially homologous to theApi m 6 protein, e.g., a protein which is different from the Api m 6protein and which is derived from the same or a different organism.Within an Api m 6 fusion protein the Api m 6 polypeptide can correspondto all or a portion of an Api m 6 protein. In a preferred embodiment, anApi m 6 fusion protein comprises at least one biologically activeportion of an Api m 6 protein. In another preferred embodiment, an Api m6 fusion protein comprises at least two biologically active portions ofan Api m 6 protein. Within the fusion protein, the term “operativelylinked” is intended to indicate that the Api m 6 polypeptide and thenon-Api m 6 polypeptide are fused in-frame to each other. The non-Api m6 polypeptide can be fused to the N-terminus or C-terminus of the Api m6 polypeptide.

[0048] For example, in one embodiment an Api m 6 fusion proteincomprises an Api m 6 T-cell proliferation-inducing domain operativelylinked to the extracellular domain of a second protein known to beinvolved in cytokine activity. Such fusion proteins can be furtherutilized in screening assays for compounds which modulate Api m 6activity.

[0049] The present invention also pertains to variants of the Api m 6proteins which function as either Api m 6 agonists (mimetics) or as Apim 6 antagonists. Variants of the Api m 6 protein can be generated bychemically synthesizing polypeptides having amino acid sequencescorresponding to the agonists or antagonists. Alternatively, the mutantscan be constructed by mutagenesis, e.g., discrete point mutation ortruncation of the Api m 6 protein. An agonist of the Api m 6 protein canretain substantially the same, or a subset, of the biological activitiesof the naturally occurring form of the Api m 6 protein. An antagonist ofthe Api m 6 protein can inhibit one or more of the activities of thenaturally occurring form of the Api m 6 protein by, for example,competitively binding to a downstream or upstream member of a cellularsignaling cascade which includes the Api m 6 protein. Thus, specificbiological effects can be elicited by treatment with a variant oflimited function. In one embodiment, treatment of a subject with avariant having a subset of the biological activities of the naturallyoccurring form of the protein has fewer side effects in a subjectrelative to treatment with the naturally occurring form of the Api m 6proteins.

[0050] For example, variants of the Api m 6 protein which function aseither Api m 6 agonists (mimetics) or as Api m 6 antagonists can beidentified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of the Api m 6 protein for Api m 6 protein agonistor antagonist activity.

[0051] Antibodies to Api m 6 Proteins

[0052] An isolated Api m 6 protein, or a portion or fragment thereof,can be used as an immunogen to generate antibodies that bind Api m 6using standard techniques for polyclonal and monoclonal antibodypreparation. A full-length Api m 6 protein can be used or,alternatively, the invention provides antigenic peptide fragments of Apim 6 for use as immunogens. The antigenic peptide of Api m 6 comprises atleast 8 amino acid residues of the amino acid sequence shown in any ofSEQ ID NOs:1-4 and encompasses an epitope of Api m 6 such that anantibody raised against the peptide forms a specific immune complex withApi m 6. Preferably, the antigenic peptide comprises at least 10 aminoacid residues, more preferably at least 15 amino acid residues, evenmore preferably at least 20 amino acid residues, and most preferably atleast 30 amino acid residues. Preferred epitopes encompassed by theantigenic peptide are regions of Api m 6 that are located on the surfaceof the protein, e.g., hydrophilic regions.

[0053] An Api m 6 immunogen can prepare antibodies as explained below inthe Examples. Alternatively, an Api m 6 immunogen is used to prepareantibodies by immunizing a suitable subject (e.g., rabbit, goat, mouseor other mammal) with the immunogen. An appropriate immunogenicpreparation can contain, for example, a chemically synthesized Api m 6polypeptide. The preparation can further include an adjuvant, such asFreund's complete or incomplete adjuvant, or similar immunostimulatoryagent. Immunization of a suitable subject with an immunogenic Api m 6preparation induces a polyclonal anti-Api m 6 antibody response.

[0054] Accordingly, another aspect of the invention pertains to anti-Apim 6 antibodies. The term “antibody” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen,such as Api m 6. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)₂ fragments which canbe generated by treating the antibody with an enzyme such as pepsin. Theinvention provides polyclonal and monoclonal antibodies that bind Api m6. The term “monoclonal antibody” or “monoclonal antibody composition”,as used herein, refers to a population of antibody molecules thatcontain only one species of an antigen binding site capable ofimmunoreacting with a particular epitope of Api m 6. A monoclonalantibody composition thus typically displays a single binding affinityfor a particular Api m 6 protein with which it immunoreacts.

[0055] Polyclonal anti-Api m 6 antibodies can be prepared as describedabove by immunizing a suitable subject with an Api m 6 immunogen. Theanti-Api m 6 antibody titer in the immunized subject can be monitoredover time by standard techniques, such as with an enzyme linkedimmunosorbent assay (ELISA) using immobilized Api m 6. If desired, theantibody molecules directed against Api m 6 can be isolated from themammal (e.g., from the blood) and further purified by well knowntechniques, such as protein A chromatography to obtain the IgG fraction.At an appropriate time after immunization, e.g., when the anti-Api m 6antibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein, Nature 256:495-497 (1975). See also, Brown et al.,J. Immunol. 127:539-46 (1981); Brown et al., J. Biol. Chem 255:498-83(1980); Yeh et al., PNAS 76:2927-31 (1976); and Yeh et al, Int. J.Cancer 29:269-75(1982). See also: the human B cell hybridoma technique(Kozbor et al., Immunol Today 4:72(1983)), the EBV-hybridoma technique(Cole et al., (1985), Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generally R.H. Kenneth, in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); Lerner, YaleJ. Biol. Med., 54:387-402 (1981); Gefter et al, Somatic Cell Genet.3:231-36(1977)). Briefly, an immortal cell line (typically a myeloma) isfused with lymphocytes (typically splenocytes) from a mammal immunizedwith an Api m 6 immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds Api m 6.

[0056] A protocol as described in the Examples below can be used toprepare monoclonal antibodies to the herein described Api m 6polypeptides. An example of such an antibody is the monoclonal antibodysecreted by hybridoma cell line 5E11. Generally, any of the many wellknown protocols used for fusing lymphocytes and immortalized cell linescan be applied for the purpose of generating an anti-Api m 6 monoclonalantibody. See, e.g., Galfre et al. Nature 266:550-552(1977). Moreover,the ordinarily skilled worker will appreciate that there are manyvariations of such methods which also would be useful. Typically, theimmortal cell line (e.g., a myeloma cell line) is derived from the samemammalian species as the lymphocytes. For example, murine hybridomas canbe made by fusing lymphocytes from a mouse immunized with an immunogenicpreparation of he present invention with an immortalized mouse cellline. Preferred immortal cell lines are mouse myeloma cell lines thatare sensitive to culture medium containing hypoxanthine, aminopterin andthymidine (“HAT medium”). Any of a number of myeloma cell lines can beused as a fusion partner according to standard techniques, e.g., theP3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. Typically,HAT-sensitive mouse myeloma cells are fused to mouse splenocytes usingpolyethylene glycol (“PEG”). Hybridoma cells resulting from the fusionare then selected using HAT medium, which kills unfused andunproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindApi m 6, e.g., using a standard ELISA assay.

[0057] Alternative to preparing monoclonal antibody-secretinghybridomas, a monoclonal anti-Api m 6 antibody can be identified andisolated by screening a recombinant combinatorial immunoglobulin library(e.g., an antibody phage display library) with Api m 6 to therebyisolate immunoglobulin library members that bind Api m 6. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit.,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, U.S. Pat No. 5,223,409;PCT International Publication Nos. WO 92/18619; WO 91/17271; WO92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; WO90/02809; and Fuchs et al, Bio/Technology 9:1370-1372 (1991); Hay et al.Hum. Antibod. Hybridomas 3:81-85 (1992); Huse et al., Science246:1275-1281 (1989); Griffiths et al, EMBO J 12:725-734 (1993); Hawkinset al., J. Mol Biol. 226:889-896 (1992); Clarkson et al., Nature352:624-628 (1991); Gram et al., PNAS 89:3576-3580 (1992); Garrad et al,Bio/Technology 9:1373-1377 (1991); Hoogenboom et al., Nuc. Acid Res.19:4133-4137 (1991); Barbas et al., PNAS 88:7978-7982 (1991); andMcCafferty et al., Nature 348:552-554 (1990).

[0058] Additionally, recombinant anti-Api m 6 antibodies, such aschimeric and humanized monoclonal antibodies, comprising both human andnon-human portions, which can be made using standard recombinant DNAtechniques, are within the scope of the invention. The term “antibody”as used herein is also intended to include chimeric, human, single chainand humanized antibodies, as well as binding fragments of suchantibodies or modified versions of such antibodies. A “chimericantibody” is intended to include an antibody in which the variableregions are from one species of animal and the constant regions are fromanother species of animal. For example, a chimeric antibody can be anantibody having variable regions which derive from a mouse monoclonalantibody and constant regions which are human. A “humanized antibody” orfragment includes any human antibody capable of retaining non-humanhypervariable regions, also termed, the complementarity-determiningregions (CDRs), for example, antibodies in which the CDRs are from onespecies of animal and the framework regions and constant regions of theantibody are from a different animal species. In a humanized antibody,the CDRs can be from a mouse monoclonal antibody and the other regionsof the antibody are human.

[0059] Chimeric mouse-human monoclonal antibodies (i.e., chimericantibodies) can be produced by recombinant DNA techniques known in theart. For example, a gene encoding the Fc constant region of a murine (orother species) monoclonal antibody molecule is digested with restrictionenzymes to remove the region encoding the murine Fc, and the equivalentportion of a gene encoding a human Fc constant region is substituted.See Robinson et al, International Patent Publication PCT/US86/02269;Akira, et al., European Patent Application 184,187; Taniguchi, EuropeanPatent Application 171,496; Morrison et al., European Patent Application173,494; Neuberger et al., International Application WO 86/01533;Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European PatentApplication 125,023; Better et al. (1988 Science 240: 1041-1043); Liu etal. (1987) PNAS 84: 3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84: 214-218; Nishimura et al., 1987,Canc. Res. 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; andShaw et al., 1988, J. Natl Cancer Inst. 80: 1553-1559.

[0060] To eliminate or minimize immune response in a subject, it isdesirable to engineer chimeric antibody derivatives, i.e., “humanized”antibody molecules that combine the non-human Fab variable regionbinding determinants with a human constant region (Fc). Such antibodiesare characterized by equivalent antigen specificity and affinity ofmonoclonal and polyclonal antibodies and are less immunogenic whenadministered to humans, and therefore more likely to be tolerated by thepatient.

[0061] A chimeric antibody can be further humanized by replacingsequences of the Fv variable region which are not directly involved inantigen binding with equivalent sequences from human Fv variableregions. General reviews of humanized chimeric antibodies are providedby Morrison, 1985, Science 229: 1202-1207 and by Oi et al., 1986,BioTechniques 4: 214. Those methods include isolating, manipulating, andexpressing the nucleic acid sequences that encode all or part ofinumunoglobulin Fv variable regions from at least one of a heavy orlight chain. Sources of such nucleic acid are well known to thoseskilled in the art and, for example, may be obtained from 7E3, ananti-GPII_(b) III_(a) antibody producing hybridoma. The recombinant DNAencoding the chimeric antibody, or fragment thereof, can then be clonedinto an appropriate expression vector. Suitable humanized antibodies canalternatively be produced by CDR substitution U.S. Pat. No. 5,225,539;Jones et al. 1986 Nature 391: 559-525; Verhoeyan et al. 1988 Science239: 1534; and Beidler et al. 1988 J. Immunol. 141: 4053-4060).

[0062] Human mAb antibodies directed against human proteins can begenerated using transgenic mice carrying the complete human immunesystem rather than the mouse system. Splenocytes from these transgenicmice immunized with the antigen of interest are used to producehybridomas that secrete human mAbs with specific affinities for epitopesfrom a human protein (see, e.g., Wood et al. International ApplicationWO 91/00906, Kucherlapati et al. PCT publication WO 91/10741; Lonberg etal. International Application WO 92/03918; Kay et al. InternationalApplication 92/03917; Lonberg, et al. 1994 Nature 368: 856-859; Green,et al. 1994 Nature Genet. 7: 13-21; Morrison, et al. 1994 Proc. Natl.Acad. Sci. USA 81: 6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90: 3720-3724; Bruggeman et al. 1991Eur J Immunol 21: 1323-1326).

[0063] Monoclonal antibodies can also be generated by other methods wellknown to those skilled in the art of recombinant DNA technology. Analternative method, referred to as the “combinatorial antibody display”method, has been developed to identify and isolate antibody fragmentshaving a particular antigen specificity, and can be utilized to producemonoclonal antibodies (for descriptions of combinatorial antibodydisplay, see, e.g., Sastry et al. 1989 PNAS 86: 5798; Huse et al. 1989Science 246: 1275; and Orlandi et al. 1989 PNAS 86: 3833). Afterimmunizing an animal with an immunogen, the antibody repertoire of theresulting B-cell pool is cloned. Methods are generally known forobtaining the DNA sequence of the variable regions of a diversepopulation by using a mixture of oligomer primers and PCR. Larrick etal., 1991, Biotechniques 11: 152-156. A similar strategy can also beenused to amplify human heavy and light chain variable regions from humanantibodies. Larrick et al., 1991, Methods: Companion to Methods inEnzymology 2: 106-110. Winter (British Patent Application NumberGB2188538A) describes a process for altering antibodies by substitutingthe complementarity determining regions (CDRs) with those from anotherspecies. The “reshaped” or “humanized” antibodies described by Winterelicit a considerably reduced immune response in humans compared tochimeric antibodies because of the considerably less murine components.Further, the half life of the altered antibodies in circulation shouldapproach that of natural human antibodies. See, also, U.S. Pat. Nos.6,111,166; 5,837,243; 6,130,364, 6,091,001, and 5,916,771, hereinincorporated by reference.

[0064] An anti-Api m 6 antibody (e.g., monoclonal antibody) can be usedto isolate Api m 6 by standard techniques, such as affinitychromatography or immunoprecipitation. An anti-Api m 6 antibody canfacilitate the purification of natural Api m 6 from a cell suspension orbee venom and of recombinantly produced Api m 6 expressed in host-cells.Moreover, an anti-Api m 6 antibody can be used to detect Api m 6 protein(e.g., in a cellular lysate or cell supernatant) in order to evaluatethe abundance and pattern of expression of the Api m 6 protein.Detection can be facilitated by coupling (i.e., physically linking) theantibody to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0065] Api m 6-Based Pharmaceutical Compositions

[0066] The Api m 6 proteins (allergens), peptides, and anti-Api m 6antibodies (also referred to herein as “active compounds”) of theinvention can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the protein, orantibody and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifingal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions. As used herein, the phrases ‘pharmaceuticalcomposition’ and ‘therapeutic composition’ are interchangeable.

[0067] Pharmaceutical compositions containing the Api m 6 proteins,peptides, or variants thereof can be administered to a mammal (such as ahuman) sensitive to Api m 6 in a form which results in a decrease in theT-cell response of the mammal upon subsequent exposure to the proteinallergen. As used herein, a decrease or modification of the T-cellresponse of a mammal sensitive to a protein allergen is defined asnon-responsiveness or diminution in symptoms to the protein allergen inthe mammal, as determined by standard clinical procedures (see e.g.,Varney, et al., British Medical Journal 302: 265-269 (1990)), includingdiminution in allergen induced asthmatic conditions. As referred toherein, a diminution in symptoms to an allergen includes any reductionin the allergic response of a mammal, such as a human, to the allergenfollowing a treatment regimen with a peptide as described herein. Thisdiminution in symptoms may be determined subjectively in a human (e.g.,the patient feels more comfortable upon exposure to the allergen), orclinically, such as with a standard skin test.

[0068] Administration of the therapeutic compositions of the presentinvention to desensitize or tolerize an individual to a protein allergenor other protein antigen can be carried out using procedures, at dosagesand for periods of time effective to reduce sensitivity (i.e., to reducethe allergic response) of the individual to a protein allergen or otherprotein antigen. Effective amounts of the therapeutic compositions willvary according to factors such as the degree of sensitivity of theindividual to the protein allergen, the age, sex, and weight of theindividual, and the ability of the peptide(s) to elicit an antigenicresponse in the individual. Dosage regimes may be adjusted to providethe optimum therapeutic response. For example, several divided doses maybe administered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

[0069] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents, antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of toxicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

[0070] Administration, e.g., subcutaneous administration, of an Api m 6protein or variant peptide as described herein to a mammal, such as ahuman, can tolerize or energize appropriate T-cell subpopulations suchthat they become unresponsive to the protein allergen and do notparticipate in stimulating an immune response upon subsequent exposure.In addition, administration of such a peptide may modify the lymphokinesecretion profile as compared with exposure to the naturally-occurringprotein allergen or portion thereof (e.g., result in a decrease of IL-4and/or an increase in IL-2). Furthermore, exposure to the peptide mayinfluence T-cell subpopulations which normally participate in theresponse to the allergen such that these T-cells are drawn away from thesite(s) of normal exposure to the allergen toward the site oftherapeutic administration of the peptide. This redistribution of T-cellsubpopulations may ameliorate or reduce the ability of an individual'simmune system to stimulate the usual immune response at the site ofnormal exposure to the allergen, resulting in a diminution in allergicsymptoms.

[0071] In addition, administration of the above-described Api m 6proteins, peptides or their variants may result in lower levels of IgEstimulation activity. Preferably, administration results in minimal IgEstimulating activity. As used herein, minimal IgE stimulating activityrefers to IgE production that is less than the amount of IgE productionand/or IL-4 production stimulated by the whole Api m 6 protein allergen.

[0072] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where the peptides or protein are watersoluble) or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersion. Forintravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). In all cases, the composition must besterile and should be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyethylene glycoland the like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmanitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

[0073] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., an Api m 6 protein, peptides or anti-Api m 6antibody) in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

[0074] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0075] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g. a gas such as carbon dioxide, or anebulizer.

[0076] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0077] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0078] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat No.4,522,811.

[0079] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0080] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0081] It is also possible to modify the structure of peptides useful inmethods of the invention for such purposes as increasing solubility,enhancing therapeutic or preventive efficacy, or stability (e.g., shelflife ex vivo, and resistance to proteolytic degradation in vivo). Amodified peptide can be produced in which the amino acid sequence hasbeen altered, such as by amino acid substitution, deletion, or addition,to modify immunogenicity and/or reduce allergenicity, or to which acomponent has been added for the same purpose. For example, the aminoacid residues essential to T-cell epitope function can be determinedusing known techniques (e.g., substitution of each residue anddetermination of presence or absence of T-cell reactivity). Thoseresidues shown to be essential can be modified (e.g., replaced byanother amino acid whose presence is shown to enhance T-cellreactivity), as can those which are not required for T-cell reactivity(e.g., by being replaced by another amino acid whose incorporationenhances T-cell reactivity but does not diminish binding to relevantMHC). Another example of a modification of peptides is substitution ofcysteine residues preferably with alanine, or glutamic acid, oralternatively with serine or threonine to minimize dimerization viadisulfide linkages.

[0082] In order to enhance stability and/or reactivity, peptides canalso be modified to incorporate one or more polymorphisms in the aminoacid sequence of a protein allergen resulting from natural allelicvariation. Additionally, D-amino acids, non-natural amino acids ornon-amino acid analogues can be substituted or added to produce amodified peptide within the scope of this invention. Furthermore,peptides can be modified to produce a peptide-PEG conjugate.Modifications of peptides can also include reduction/alkylation (Tarrin: Methods of Protein Microcharacterization, J. E. Silver, ed HumanaPress, Clifton, N.J., pp 155-194 (1986)); acylation (Tarr, supra);esterification (Tarr, supra); chemical coupling to an appropriatecarrier (Mishell and Shiigi, eds., Selected Methods in CellularImmunology, W H Freeman, San Francisco, Calif. (1980); U.S. Pat. No.4,939,239); or mild formalin treatment (Marsh International Archives ofAllergy and Applied Immunology 41: 199-215 (1971)).

[0083] To facilitate purification and potentially increase solubility ofpeptides, it is possible to add reporter group(s) to the peptidebackbone. For example, poly-histidine can be added to a peptide topurify the peptide on immobilized metal ion affinity chromatography.Hochuli, et al., Bio/Technology, 6:1321-1235 (1988). In addition,specific endoprotease cleavage sites can be introduced, if desired,between a reporter group and amino acid sequences of a peptide tofacilitate isolation of peptides free of irrelevant sequences. In orderto successfully desensitize an individual to a protein antigen, it maybe necessary to increase the solubility of a peptide by addingfunctional groups to the peptide or by not including hydrophobic T-cellepitopes or regions containing hydrophobic epitopes in the peptide.

[0084] To potentially aid proper antigen processing of T-cell epitopeswithin a peptide, canonical protease sensitive sites can berecombinantly or synthetically engineered between regions, eachcomprising at least one T-cell epitope. For example, charged amino acidpairs, such as KK or RR, can be introduced between regions within apeptide during synthesis.

[0085] The invention further encompasses at least one therapeuticcomposition useful in treating a disease which involves an immuneresponse to protein antigen (e.g., an allergen, an autoantigen, etc.)comprising at least one peptide having a sufficient percentage of theT-cell epitopes of the protein antigen such that in a substantialpercentage of a population of individuals sensitive to the proteinantigen, the response of such individuals to the protein antigen issubstantially diminished, with the provision that the at least onepeptide does not comprise the entire protein antigen.

[0086] Kits Including Api m 6 Proteins or Antibodies

[0087] The present invention additionally provides kits for diagnosticuse that are comprised of one or more containers containing an Api m 6protein or Api m 6 antibody and, optionally, a labeled binding partnerto the antibody. The label incorporated into the antibody may include,but is not limited to, a chemiluminescent, enzymatic, fluorescent,colorimetric or radioactive moiety. The kit may, optionally, furthercomprise a predetermined amount of a purified aforementioned peptide, ornucleic acids thereof, for use as a diagnostic, standard, or control inthe aforementioned assays.

EXAMPLES

[0088] The invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims. The following examples illustrate the identification,characterization and applications of Api m 6 protein.

Example 1 Purification of Api m 6 Isoforms

[0089] The Api m 6 protein was identified in studies examining thereactivity of IgE-sera derived from patients hypersensitive to purifiedbee venom (BV) proteins.

[0090] Serum and peripheral blood mononuclear cells (PBMC) were obtainedfrom BV hypersensitive patients (grade II-IV, according to Mueller'sclassification). Müeller, J Asthma Res 3: 331-333 (1966). All patientshad BV specific IgE (≧0.35 kU/l; CAP® system Pharmacia, Uppsala, Sweden)and positive intradermal skin tests (≧0.1 μg/ml, Pharmalgen®, ALK,Hørsholm, Denmark).

[0091] BV proteins were separated by 15% SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) under non-reducingconditions and blotted to PVDF membranes in CAPS/methanol buffer (10 mMCAPS, 10% methanol, pH 11). Membranes were blocked with non-fat milk(5%) in phosphate buffered salt solution containing 0.1% Tween 20(PBS-Tween), then incubated with patients' sera ({fraction (1/10)} inPBS-Tween) for 24 h at 4° C. Specific IgE binding was detected using abiotinylated monoclonal mouse anti-human IgE antibody (Pharmingen,Hamburg, Germany) followed by incubation with streptavidin conjugatedhorseradish peroxidase (HRP) (UBI, Lucema Chem AG, Luzern, Switzerland).Peroxidase reactivity was visualized by enhanced chemiluminescence (ECL,Amersham, UK).

[0092] Analysis of IgE sera from 43 patients reactive with separated BVproteins revealed a previously undescribed band at about 8 kD in 18(42%) of the samples. The 8 kDa protein corresponding to the observed 8kDa band was purified from other BV proteins by size exclusionchromatography. Chromatography was performed by lyophilizing whole BV(Apis mellifera) (Latoxan, Rosans, France) in 50% formic acid. Particleswere removed by centrifugation and filtration prior to sampleapplication to a BioRad P-60 column (2.5×100 cm) (BioRad, Glattbrugg,Switzerland) equilibrated in 50% formic acid. Acidic conditions wereused to minimize melittin tetramer formation. Bello, et al.,Biochemistry 21: 461-465 (1982). Fractions of 4 ml were collected at aflow rate of 6.5 ml/h. Each fraction was lyophilized, dissolved in 0.02N acetic acid and analyzed by SDS-PAGE. Laemmili, Nature 227: 680-685(1970). Fractions containing the 8 kDa band eluted in a broad peakbetween the peaks of two other bee venom proteins, PLA₂ and melittin.MALDI-TOF mass spectrometry analysis of these fractions revealed thepresence of four proteins with molecular weights of 7,190, 7400, 7,598,and 7,808 Da.

[0093] The four proteins were further purified by reverse phase HPLCusing two runs through a C₄ column (Phenomex W-Porex 5; 250×46 mm;Rancho Palos Verdes, Calif., USA). Water-acetonitrile gradient was usedfor separation (buffer A: 10% acetonitrile, 0.1% trifluoroacetic acid inwater; buffer B: 90% acetonitrile, 0.1% trifluoroacetic acid in water).All four proteins were recognized by IgE from a BV hypersensitivepatient that was positive for the 8 kDa protein in the initialscreening. The four proteins were named Api m 6.01, Api m 6.02, and Apim 6.03, and Api m 6.04, respectively.

Example 2 Elucidation of the Amino Acid Sequence of Api m 6 Isoforms

[0094] The amino acid sequence of the 8 kDa protein isoforms wasdetermined by two approaches: N-terminal sequence analysis by Edmandegradation and C-terminal sequencing using carboxypeptidases incombination with mass spectrometry.

[0095] Amino-terminal sequence analysis of proteins and proteolyticfragments was carried out with a pulse-liquid phase microsequencer,model 477A (Applied Biosystems, Foster City, Calif.) using standardprograms. Proteins were reduced (8 M Urea, 0.15 M Tris-HCl, 2.5 mM1,4-dithiothreitol (DTT), pH 8.6) and alkylated (7.5 mM sodiumiodoacetate) prior to desalting on a C₈ reverse phase column. Alkylated7.6 kD (Api m 6.03) protein was incubated with trypsin (sequencinggrade, Boehringer Mannheim AG, Rotkreuz, Switzerland) overnight at roomtemperature (RT) (50 mM Tris-H.Cl, pH 8.6); fragments were separated byHPLC (C₈ column 5 μm HAIsil™, 2.1×100 mm; Higgins Analytical Inc.).

[0096] Carboxyl terminal sequence analysis of proteins and proteolyticfragments was performed by matrix-assisted laser desorption-ionizationtime of flight (MALDI-TOF) mass spectrometry, on a voyager-DE™RP(PerSeptive Biosystems, Framingham, Mass., USA). Patterson, et al., AnalChem 67: 3971-3978 (1995). V8 proteinase (Endoproteinase Glu-C) waspurchased from Promega (Zurich, Switzerland) and used according to themanufacturer's instructions. Endoproteinase Arg-C sequencing grade,endoproteinase Asp-N sequencing grade, carboxypeptidase Y sequencinggrade and carboxypeptidase A were purchased from Boehringer Mannheim AG(Rotkreuz, Switzerland). Enzymatic fragmentation with Arg-C was carriedout at either room temperature (RT) or 4° C. in 15 mM HEPES buffer (pH8) with 10 mM DTT. The enzyme to protein-ratio was {fraction (1/50)}(w/w). The reaction was stopped by adding matrix solution (saturatedsolution of sinapinic acid, 10 mg/ml, in acetonitrile/water 30/70%(v/v)). Fragments from Asp-N digestion (enzyme to protein ratio of{fraction (1/125)} (w/w) in 15 mM ammonium acetate buffer, pH 6.5) ofreduced proteins (2 mM DTT in water, 37° C. overnight) were separated byHPLC for further analysis. Determination of free SH-groups of cysteineswas done by incubation with N-ethylmaleimide (NEM). C-terminal aminoacids were determined by incubation of proteins with carboxypeptidase A(enzyme to protein ratio of {fraction (1/10)} to {fraction (1/100)}(w/w) in 15 mM HEPES buffer, pH 7.5) or carboxypeptidase Y (enzyme toprotein ratio of {fraction (1/10)} to {fraction (4/100)} (w/w) in, 15 mMammonium acetate, pH 6) at 4° C. or room temperature. Experimentalconditions were optimized for each substrate preparation. The reactionwas stopped by the addition of matrix solution.

[0097] The first 48 amino acids were resolved by direct sequencing ofthe reduced and alkylated protein. Overlapping internal segments wereobtained by sequencing of HPLC purified tryptic peptides. C-terminalsequence, on the other hand, could only be determined by sequencingusing carboxypeptidases. C-terminal residues were resolved inindependent experiments using either carboxypeptidase Y or A. Longstretches of sequence data obtained by N-terminal analysis werefurthermore confirmed by C-terminal analysis. The amino acid sequencesof the Api m 6 isoforms is shown in Table 1. TABLE 1 Amino AcidSequences of Api m 6.01, 6.02, 6.03, and 6.04GGFGGLGGRGKCPSNEIFSRCDGRCQRFCPNVVPKPLCIKICAPGCVCRLGYLRNKKKVCVPRSKCG (SEQID NO:1)GGFGGLGGRGKCPSNEIFSRCDGRCQRFCPNVVPKPLCIKICAPGCVCRLGYLRNKKKVCVPRSKCG(P,L)(SEQ ID NO:2)FGGFGGFGGLGGRGKCPSNEIFSRCDGRCQRFCPNVVPKPLCIKICAPGCVCRLGYLRNKKKVCVPRSKCG(SEQ ID NO:3)FGGFGGFGGLGGRGKCPSNEIFSRCDGRCQRFCPNVVPKPLCIKICAPGCVCRLGYLRNKKKVCVPRSKCG(P,L)(SEQ ID NO:4)

[0098] Api m 6.03 was further analyzed by mass spectrometry afterincubation with either endoproteinase Arg-C, Asp-N or V8. Signals forproteolytic peptides were consistent with theoretical mass of expectedfragments and confirmed the positions of arginine, aspartate andglutamate residues.

[0099] EMBL and SWISS PROT data base searches for protein sequencehomology and computer assisted protein analysis were done applying theWisconsin Package Version 9.1 software (Genetics Computer Group,Madison, Wis., USA). Database searches revealed that Api m 6 containedan epidermal growth factor-like domain signature, which is shared bymany otherwise unrelated proteins. See Davis, New Biol 2:410-419 (1990).No obvious homology to known proteins was found even when a profilesearch with the particular cysteine spacing motif “CX₈CX₃CX₃CX₈CX₃CX₃C”(where X is any amino acid) was performed.

Example 3 Generation of Antibodies to Api m 6 Isoforms

[0100] A B-cell hybridoma line producing monoclonal anti-Api m 6.03 (SEQID NO:3) antibodies was established from mice immunized with Api m 6.03(SEQ ID NO:3). The hybridoma culture supernatant was diluted 1:25,000 inPBS-Tween 1% milk and incubated with membranes for 1 hour at RT.Specific antibody binding was detected with HRP conjugated sheepanti-mouse Ig antibody (Amershaam, UK) and peroxidase reactivity wasvisualized by enhanced chemiluminescence.

Equivalents

[0101] From the foregoing detailed description of the specificembodiments of the invention, it should be apparent that a novel beevenom allergen has been described. Although particular embodiments havebeen disclosed herein in detail, this has been done by way of examplefor purposes of illustration only, and is not intended to be limitingwith respect to the scope of the appended claims which follow

1 5 1 67 PRT Apis mellifera 1 Gly Gly Phe Gly Gly Leu Gly Gly Arg GlyLys Cys Pro Ser Asn Glu 1 5 10 15 Ile Phe Ser Arg Cys Asp Gly Arg CysGln Arg Phe Cys Pro Asn Val 20 25 30 Val Pro Lys Pro Leu Cys Ile Lys IleCys Ala Pro Gly Cys Val Cys 35 40 45 Arg Leu Gly Tyr Leu Arg Asn Lys LysLys Val Cys Val Pro Arg Ser 50 55 60 Lys Cys Gly 65 2 69 PRT Apismellifera Where XX at position 68 and 69 is either PL or LP. 2 Gly GlyPhe Gly Gly Leu Gly Gly Arg Gly Lys Cys Pro Ser Asn Glu 1 5 10 15 IlePhe Ser Arg Cys Asp Gly Arg Cys Gln Arg Phe Cys Pro Asn Val 20 25 30 ValPro Lys Pro Leu Cys Ile Lys Ile Cys Ala Pro Gly Cys Val Cys 35 40 45 ArgLeu Gly Tyr Leu Arg Asn Lys Lys Lys Val Cys Val Pro Arg Ser 50 55 60 LysCys Gly Xaa Xaa 65 3 71 PRT Apis mellifera 3 Phe Gly Gly Phe Gly Gly PheGly Gly Leu Gly Gly Arg Gly Lys Cys 1 5 10 15 Pro Ser Asn Glu Ile PheSer Arg Cys Asp Gly Arg Cys Gln Arg Phe 20 25 30 Cys Pro Asn Val Val ProLys Pro Leu Cys Ile Lys Ile Cys Ala Pro 35 40 45 Gly Cys Val Cys Arg LeuGly Tyr Leu Arg Asn Lys Lys Lys Val Cys 50 55 60 Val Pro Arg Ser Lys CysGly 65 70 4 73 PRT Apis mellifera Where XX at position 72 and 73 iseither PL or LP. 4 Phe Gly Gly Phe Gly Gly Phe Gly Gly Leu Gly Gly ArgGly Lys Cys 1 5 10 15 Pro Ser Asn Glu Ile Phe Ser Arg Cys Asp Gly ArgCys Gln Arg Phe 20 25 30 Cys Pro Asn Val Val Pro Lys Pro Leu Cys Ile LysIle Cys Ala Pro 35 40 45 Gly Cys Val Cys Arg Leu Gly Tyr Leu Arg Asn LysLys Lys Val Cys 50 55 60 Val Pro Arg Ser Lys Cys Gly Xaa Xaa 65 70 5 35PRT Artificial Sequence Description of Artificial Sequence CYSTEINESPACING MOTIF 5 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa CysXaa Xaa 1 5 10 15 Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa XaaXaa Cys Xaa 20 25 30 Xaa Xaa Cys 35

What is claimed is:
 1. A substantially pure polypeptide comprising anamino acid sequence at least 70% identical to the amino acid sequence ofSEQ ID NO:1
 2. The polypeptide of claim 1, wherein said polypeptidecomprises an amino acid sequence at least 90% identical to the aminoacid sequence of SEQ ID NO:
 1. 3. The polypeptide of claim 1, whereinsaid polypeptide comprises the amino acid sequence of SEQ ID NO:
 1. 4.The polypeptide of claim 1, wherein said polypeptide comprises an aminoacid sequence at least 70% identical to the amino acid sequence of SEQID NO:
 2. 5. The polypeptide of claim 1, wherein said polypeptidecomprises an amino acid sequence at least 70% identical to the aminoacid sequence of SEQ ID NO:
 3. 6. The polypeptide of claim 1, whereinsaid polypeptide comprises an amino acid sequence at least 70% identicalto the amino acid sequence of SEQ ID NO:
 4. 7. The polypeptide of claim1, wherein said polypeptide is an Apis mellifera bee venom protein. 8.The polypeptide of claim 1, wherein said polypeptide is glycosylated. 9.The polypeptide of claim 1, wherein said polypeptide binds to a humanIgE antibody.
 10. The polypeptide of claim 1, wherein said polypeptidestimulates T-cell proliferation.
 11. The polypeptide of claim 1, whereinsaid polypeptide binds to the monoclonal antibody 5E11 (AccessionNo.______).
 12. An antibody which binds to the polypeptide of claim 1.13. The antibody of claim 12, wherein said antibody is a monoclonalantibody.
 14. The antibody of claim 12, wherein said antibody is apolyclonal antibody.
 15. The antibody of claim 12, wherein said antibodyis a humanized antibody.
 16. The antibody of claim 13, wherein saidantibody binds to the same epitope to which the monoclonal antibodyproduced by the hybridoma 5E11 (Accession No______) binds.
 17. Theantibody of claim 16, wherein said antibody is the antibody produced bythe hybridoma 5E11 (Accession No.______).
 18. A hybridoma producing anantibody which binds to the same epitope to which the monoclonalantibody produced by the 5E11 (Accession No. ______) binds.
 19. Thehybridoma of claim 18, wherein said hybridoma is hybridoma 5E11(Accession No. ______).
 20. A composition comprising polypeptidefragments of the Api m 6 protein, wherein said polypeptide fragments arebetween 6-72 amino acids in length.
 21. The composition of claim 20,wherein said polypeptide fragments are between 20-100 amino acids inlength.
 22. The composition of claim 20, wherein said polypeptidefragments are between 30-70 amino acids in length.
 23. The compositionof claim 20, wherein said polypeptide fragments are between 40-60 aminoacids in length
 24. The composition of claim 20, wherein at least onepolypeptide in the composition has an amino acid sequence that overlapsby at least 3 amino acids with at least one other polypeptide in thecomposition
 25. The composition of claim 20, wherein the polypeptidefragments of Api m 6 overlap by between 5 and 10 amino acids.
 26. Thecomposition of claim 20, wherein the composition comprises of a set ofpolypeptide fragments that map the total length of the Api m 6 protein.27. A pharmaceutical composition comprising the polypeptide of claim 1and a pharmaceutically acceptable carrier.
 28. The pharmaceuticalcomposition of claim 27, further comprising a second bee venompolypeptide.
 29. The pharmaceutical composition of claim 28, whereinsaid second bee venom polypeptide is selected from the group comprisingphospholipase A₂, hyaluronidase, allergen C, mellitin, adolapin,minimine, acid phosphatase, protease inhibitor, and glycosylatedIgE-binding proteins, or analogs or derivatives thereof.
 30. A method ofmodulating an immune response, said method comprising administering thepolypeptide of claim 1 to a subject in need thereof in an amountsufficient to inhibit an immune reaction by the subject against saidpolypeptide.
 31. The method of claim 30, further comprisingadministering a second bee venom polypeptide to said subject.
 32. Themethod of claim 31, wherein the second bee venom polypeptide isphospholipase A₂, hyaluronidase, allergen C, mellitin, adolapin,minimine, acid phosphatase, protease inhibitor, and acid phosphatase,and glycosylated IgE-binding proteins, or analogs or derivativesthereof.
 33. A method of identifying an individual at risk for bee venomhypersensitivity, the method comprising: administering to saidindividual the polypeptide of claim 1; and measuring an immune responseraised against said polypeptide, wherein a detectable immune responseindicates that said individual is at risk for bee venomhypersensitivity.
 34. The method of claim 33, wherein said polypeptideis administered to said subject intradermally.
 35. The method of claim34, wherein said polypeptide is administered at a concentration of lessthan about 1 μg/ml.
 36. A method of purifying the polypeptide of claim1, the method comprising: providing a cell expressing the polypeptide ofclaim 1; contacting said cell with an antibody which binds topolypeptide comprising an amino acid sequence at least 70% identical tothe amino acid sequence of SEQ ID NO:1 to form a polypeptide-antibodycomplex; isolating said antibody-polypeptide complex; and recoveringsaid polypeptide from said antibody-polypeptide complex, therebypurifying said polypeptide.
 37. A kit comprising, in one or morecontainers, a substance selected from the group consisting of: an Api m6 polypeptide, a overlapping polypeptide fragments of an Api m 6polypeptide, an antibody against said Api m 6 polypeptide.